The present invention relates to the 190 Kd neuronal protein (hereinafter xe2x80x9cp190xe2x80x9d, xe2x80x9cCASPRxe2x80x9d or xe2x80x9cCASPR/p190xe2x80x9d) that interacts with contactin, and with the carbonic anhydrase (xe2x80x9cCAHxe2x80x9d) domain of the receptor-type tyrosine phosphatase RPTP-xcex2, specific peptides thereof and nucleic acid molecules encoding such p190 proteins and peptides. The protein is also referred to as CASPR, for Contactin ASsociated PRotein. The CAH domain of RPTPxcex2 has previously been identified as a ligand for contactin, and the binding of the CAH domain of RPTPxcex2 to the contactin on neural cells results in neurite growth, differentiation and survival. CASPR/p190 has been identified as a potential bridge that couples contactin, a GPI-linked protein, with intracellular second messenger systems. The invention also relates to compounds that mimic, enhance, or suppress the effects of p190, including those molecules which act downstream in the signal transduction pathway that results from the binding of the ligand to contactin. In addition, the invention also relates to the use of such compounds to treat neurologic diseases including those characterized by insufficient, aberrant, or excessive neurite growth, differentiation or survival.
The ability of cells to respond to signals from their microenvironment is a fundamental feature of development. In the developing nervous system, neurons migrate and extend axons to establish their intricate network of synaptic connections (Goodman and Shatz, 1993, Cell/Neuron (Suppl.), 72/10:77-98). During migration and axonal pathfinding, cells are guided by both attractive and repulsive signals (Hynes and Lander, 1992, Cell, 68:303-322; Keynes and Cook, 1992, Lurr. Opin. Neurobiol., 2:55-59). The ability of the neuron to respond to these signals requires cell surface molecules that are able to receive the signal and to transmit it to the cell interior resulting in specific biological responses.
It is well established that protein tyrosine phosphorylation is responsible for the regulation of many cellular responses to external stimuli crucial for cell growth, proliferation and differentiation (Schlessinger and Ullrich, 1992, Neuron, 9:383-391). Tyrosine phosphorylation has been implicated in several developmental processes in the nervous system. For example, receptor tyrosine kinases were shown to effect neuronal survival (Chao, 1992, Neuron, 9:583-593), and cell fate determination (Zipursky and Rubin, 1994, Annu. Rev. Neurosci., 17:373-397). Non-receptor tyrosine kinases have been shown to be downstream elements in signaling via cell recognition molecules that play a role in cell guidance and migration (Ignelzi et al., 1994, Neuron, 12:873-884; Umemori et al., 1994, Nature, 367-572-586).
The transient nature of signaling by phosphorylation requires specific phosphatases for control and regulation (Hunter, 1995, Cell, 80:225-236). Indeed, many protein tyrosine phosphatases have been shown to be expressed in specific regions of the developing brain, including the olfactory neuroepithelium (Walton et al., 1993, Neuron, 11:387-400), the cortex (Sahin et al., 1995, J. Comp. Neurol., 351:617-631), and in retinal Mxc3xcller glia (Shock et al., 1995, Mol. Brain Res., 28:110-116). Furthermore, expression of several tyrosine phosphatases, such as PTPxcex1 (den Hertog et al., 1993, EMBO J., 12:3789-3798), PC12-PTP1 (Sharama and Lombroso, 1995, J. Biol. Chem., 270:49-53) and several forms of LAR (Zhang and Longo, 1995, J. Cell. Biol., 128:415-431) have been found to be regulated during neural differentiation of P19 or PC12 cells.
Receptor-type tyrosine phosphatases (RPTPs) have been subdivided into several groups based on structural characteristics of their extracellular domains (Charbonneau and Tonks, 1992, Annu. Rev. Cell Biol., 8:463-493; Barnea et al., 1993, Mol. Cell. Biol., 13:1497-1506). RPTPxcex2/xcex6 and RPTPxcex3 are members of a distinct group of phosphatases, characterized by the presence of a carbonic anhydrase-like domains (CAH), fibronectin type III repeats (FNIII), and a long cysteine free region (spacer domain) in their extracellular domain (Barnea et al., 1993, Mol. Cell. Biol., 13:1497-1506; Krueger et al., 1992, Proc. Natl. Acad. Sci. USA, 89:7417-7421; Levy et al., 1993, J. Biol. Chem., 268:10573-10581). The expression of RPTPxcex2 is restricted to the central and peripheral nervous system, while RPTPxcex3 is expressed both in the developing nervous system, as well as, in a variety of other tissues in adult rat (Canoll et al., 1993, Dev. Brain Res., 75:293-298; Barnea et al., 1993, Mol. Cell. Biol., 13:1497-1506). RPTPxcex2 exists in three forms, one secreted form and two membrane bound forms, that differ by the absence of 860 residues from the spacer domain (Levy et al., 1993, J. Biol. Chem., 268:1053-10582; Maurel et al., 1994, Proc. Natl. Acad. Sci. USA, 91:2512-2516). The secreted form has been identified as a chondroitin sulfate proteoglycan from rat brain called phosphocan (3F8 proteoglycan) (Barnea et al., 1994, Cell, 76:205; Maurel et al., 1994, Proc. Natl. Acad. Sci. USA, 91:2512-2516; Shitara et al., 1994, J. Biol. Chem. 269:20189-20193). The transmembrane form has also been shown to be expressed in a form of a chondroitin sulfate proteoglycan (Barnea et al., 1994, J. Biol. Chem., 269:14349-14352). Purified phosphocan can interact in vitro with the extracellular matrix protein tenascin, and with the adhesion molecules, N-CAM and Ng-CAM (Barnea et al., 1994, J. Biol. Chem., 269:14349-14352; Grumet et al., 1993, J. Cell. Biol., 120:815-824; Grumet et al., 1994, J. Biol. Chem., 269:12142-12146; Milev et al., 1994, J. Cell. Biol., 127:2512-2516).
The present invention relates to the 190 Kd neuronal protein (hereinafter xe2x80x9cp190xe2x80x9d, xe2x80x9cCASPRxe2x80x9d or xe2x80x9cCASPR/p190xe2x80x9d) that interacts with contactin, and with the carbonic anhydrase (xe2x80x9cCAHxe2x80x9d) domain of the receptor-type tyrosine phosphatase RPTP-xcex2, specific peptides thereof and nucleic acid molecules encoding such p190 proteins and peptides.
The invention further relates to the use of p190 and related compounds to treat neurologic diseases including those characterized by insufficient, aberrant, or excessive neurite growth, differentiation or survival. More specifically, the invention relates to the use of compounds that mimic, enhance or suppress the effects of p190 on neurite growth, differentiation and survival.
The invention is based, in part, on the discovery that the CAH domain of RPTPxcex2 (RPTPxcex2-CAH) is the ligand for contactin and that its binding results in neurite growth, differentiation and survival, and on the further discovery that p190 acts as the bridge between contactin and intracellular second messenger systems.
In the examples described infra, it is shown that receptor phosphatase RPTPxcex2 specifically interacts with two ligands, one on the surface of glial cells, and the other on the surface of neuronal cells. Using expression cloning in COS7 cells and bioaffinity purification, the neuronal ligand was identified to be the rat homologue of the cell recognition molecule contactin (F11/F3). Using combinations of soluble and membrane bound forms of RPTPxcex2 and contactin it is demonstrated that the reciprocal interaction between the two molecules is mediated by the CAH domain of the phosphatase. Moreover, it is found that when used as a substrate, the CAH domain of RPTPxcex2 induced neurite growth, differentiation and survival of primary neurons and IMR-32 neuroblastoma cells. Using antibody perturbation experiments, the contactin ligand was found to be a neuronal receptor for the CAH domain of RPTPxcex2. The data indicate that the interactions between contactin, a cell recognition molecule, and RPTBxcex2, a transmembrane protein tyrosine phosphatase, plays an important role in neuronal development and differentiation. As explained more fully in Section 5.2, the further experiments of the examples were conducted to elucidate the interaction between contactin and intracellular second messenger systems. Binding experiments revealed that the interaction between p190 and contactin is important for the role of contactin and RPTPxcex2-CAH in neuronal growth, development and differentiation.
3.1. DEFINITIONS
As used herein, the following terms and abbreviations shall have the meanings indicated below:
As used herein, the word xe2x80x9cmodulatexe2x80x9d shall have its usual meaning, but shall also encompass the meanings of the words enhance, inhibit, and mimic. In addition, as used herein, the word xe2x80x9cexpressionxe2x80x9d, when used in connection with a gene such as p190, shall have its usual meaning, but shall also encompass the transcription of the gene, the longevity of the functional mRNA transcribed from the gene, the translation of that mRNA, and the activity of the gene product.